This study was designed to determine the source of tumor necrosis factor (TNF) alpha within the porcine corpus luteum (CL). 1) Sections of frozen or paraffin-embedded CL from various stages of the estrous cycle were incubated with the following primary antibodies: anti-human recombinant TNFalpha, anti-porcine macrophage-specific antigen, or anti-alpha-actin (marker of pericyte and smooth muscle cells). Dolichos biflorus lectin-peroxidase was used as an endothelial cell label. Positive immunostaining for TNFalpha was apparent in porcine CL throughout the estrous cycle. TNFalpha immunoreactivity was primarily localized in cells along septal/vascular tracts, and exhibited spatial and temporal distribution similar to that of cells labeled with anti-macrophage antibodies. Large luteal cells exhibited weak staining for TNFalpha in paraffin sections, whereas microvascular endothelial cells were consistently negative in both frozen and paraffin sections. 2) Enriched subpopulations of macrophages, endothelial cells, and large and small luteal cells were isolated by density gradient and immunomagnetic bead separation techniques. TNFalpha secretion by each subpopulation was determined by measuring bioactive TNFalpha in incubation media using a specific in vitro bioassay. Macrophage subpopulations secreted up to 100-fold greater quantities of bioactive TNFalpha (up to 400 pg/10(6) cells) than did other subpopulations. In contrast, endothelial cell and small luteal cell subpopulations released very small amounts (< 8 pg/10(6) cells) of bioactive TNFalpha. Large luteal cells secreted slightly greater amounts of TNFalpha (10-15 pg/10(6) cells). Local macrophages appear to be the primary source of TNFalpha in the porcine CL.